告达庭抑制DNA损伤修复与糖酵解途径增加乳腺癌放疗敏感性的机制研究

Radioresistance is the main cause of radiotherapy failure. DNA damage repair and the change of tumor metabolism are primary factors leading to radioresistance. Our recently studies showed that caudatin, a C21 steroidal aglycone isolated from Cynanchum bungei, could induce DNA damage and increase the expression of γ-H2AX, a marker of DNA damage. Moreover, caudatin treatment inhibited the production of lactate and downregulated the expression of HK-2 in breast cancer cells. Based on the findings of caudatin increasing the radiosensitivity of breast cancer, we mainly investigate the molecular mechanisms that caudatin sensitizes breast cancer cells to radiation therapy by inhibiting the DNA damage repair and aerobic glycolysis in this project. We take combinatory approaches of a variety of experimental techniques, such as western blotting, immunofluorescence technique and assays of biological response to ionizing radiation to explore the molecular mechanisms that caudatin enhances radiosensitivity through regulating DNA damage response and aerobic glycolysis in human breast cancer cells. Implementation of the project will illustrate the anti-tumor mechanisms for caudatin, and provide theoretical base for exploiting the resource of traditional Chinese herbal medicine and human breast cancer therapy.

肿瘤细胞的辐射抵抗是导致放疗失败的主要原因，而DNA损伤修复与肿瘤细胞代谢的改变则是决定肿瘤放疗抵抗的重要因素。我们近期研究发现，从泰山白首乌中分离得到的C21甾体化合物-告达庭能够诱导乳腺癌细胞DNA发生断裂，并且上调DNA损伤标志分子γ-H2AX的表达。同时，告达庭能够抑制细胞内乳酸生成和己糖激酶 (HK-2) 的表达。本项目拟在证实告达庭对乳腺癌具有放疗增敏作用的基础上，从抑制DNA损伤修复和调节肿瘤细胞糖酵解途径等方面阐明告达庭降低乳腺癌放疗抵抗的分子机理。综合运用免疫印迹、免疫荧光与辐射细胞生物学效应检测等研究手段，阐明告达庭调控肿瘤细胞DNA损伤应答和糖类代谢与其增加乳腺癌放射敏感性的内在联系。项目的实施在丰富告达庭抑癌活性分子机理的同时，为中药资源开发利用及乳腺癌临床治疗提供新思路和理论指导。

本项目主要探讨白首乌中的C21甾体苷元-告达庭抗肿瘤活性的分子机制。在确定告达庭诱导肿瘤细胞DNA损伤应答的基础上，阐明其增强乳腺癌辐射治疗（IR）和TRAIL敏感性的分子机理。我们研究发现，告达庭能够剂量依赖性地抑制乳腺癌细胞活力，并通过诱导细胞发生G0/G1期阻滞而抑制细胞增殖，同时上调p21和p27的表达。乳腺癌细胞经告达庭单独处理后，发生明显的DNA损伤和凋亡现象，表现为γ-H2AX表达水平上调，CHK2/ATM通路被激活，同时caspase家族蛋白切割水平增加。此外，告达庭能够抑制DNA损伤修复相关蛋白如Rad51的表达，增强乳腺癌细胞的放疗敏感性。告达庭作用乳腺癌细胞后，内质网应激和MAPK通路被激活，死亡受体DR5表达水平上调，从而增强TRAIL诱导的乳腺癌细胞凋亡；敲低DR5表达则抑制告达庭对乳腺癌细胞TRAIL的增敏作用。此外，告达庭处理乳腺癌细胞后，LC3-II表达水平和细胞内斑点数量增加；抑制自噬则促进了告达庭诱导的DNA双链断裂和细胞凋亡，表明告达庭诱导乳腺癌细胞发生保护性自噬。HDAC/PI3K双靶点抑制剂CUDC-907同样能够促进TRAIL诱导的乳腺癌细胞凋亡。CUDC-907能够下调XIAP等凋亡抑制蛋白的表达，同时通过激活JNK而上调DR5蛋白水平；siRNA敲低DR5的表达则显著拮抗CUDC-907与TRAIL联用诱导的细胞凋亡。另外，半枝莲碱A（SBT-A）能够明显抑制肝癌细胞增殖并诱导细胞凋亡，SBT-A抗肿瘤活性与其激活内质网应激和MAPK信号通路相关。SBT-A能够明显诱导MCF-7乳腺癌细胞发生DNA双链断裂。